EP1260848B1 - Dispositif pour la détermination d'une efficacité lumineuse, microscope et méthode de microscopie - Google Patents

Dispositif pour la détermination d'une efficacité lumineuse, microscope et méthode de microscopie Download PDF

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Publication number
EP1260848B1
EP1260848B1 EP02100561A EP02100561A EP1260848B1 EP 1260848 B1 EP1260848 B1 EP 1260848B1 EP 02100561 A EP02100561 A EP 02100561A EP 02100561 A EP02100561 A EP 02100561A EP 1260848 B1 EP1260848 B1 EP 1260848B1
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EP
European Patent Office
Prior art keywords
light
detector
beam splitter
microscope
illuminating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP02100561A
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German (de)
English (en)
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EP1260848A3 (fr
EP1260848A2 (fr
Inventor
Heinrich Ulrich
Holger Dr. Birk
William Hay
Holger Nissle
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Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems Heidelberg GmbH
Leica Microsystems CMS GmbH
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Publication of EP1260848A3 publication Critical patent/EP1260848A3/fr
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/108—Beam splitting or combining systems for sampling a portion of a beam or combining a small beam in a larger one, e.g. wherein the area ratio or power ratio of the divided beams significantly differs from unity, without spectral selectivity
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/144—Beam splitting or combining systems operating by reflection only using partially transparent surfaces without spectral selectivity
    • G—PHYSICS
    • G11—INFORMATION STORAGE
    • G11B—INFORMATION STORAGE BASED ON RELATIVE MOVEMENT BETWEEN RECORD CARRIER AND TRANSDUCER
    • G11B7/00—Recording or reproducing by optical means, e.g. recording using a thermal beam of optical radiation by modifying optical properties or the physical structure, reproducing using an optical beam at lower power by sensing optical properties; Record carriers therefor

Definitions

  • the invention relates to a device for determining the light output of a Light beam with a beam splitter and one assigned to the beam splitter Detector) whereby the beam splitter splits measurement light from the light beam and feeds the detector and wherein the beam splitter and the detector become one Unit are summarized.
  • the invention also relates to a microscope with a light source that has a Illuminating light beam emitted for illuminating a sample, with at least a detector device for detecting that emanating from the sample Detection light (61) and a device for determining the light output an illuminating light beam with a beam splitter and a dem Beam splitter associated detector, the beam splitter measuring light from the The illuminating light beam (37) is split off and fed to the detector (11).
  • the invention also relates to a method for microscopy.
  • the device includes a beam splitter that emits measuring light the light emitted by the semiconductor laser splits off and one immediately detector arranged on the beam splitter, which receives the measuring light.
  • Arrangements of the type mentioned achieve only limited accuracy and reproducibility. Among other things, they are susceptible to external disturbances, in particular to vibrations and thermal influences.
  • a sample is illuminated with a light beam in order to the detection light emitted by the sample, as reflection or Fluorescent light, to observe.
  • the focus of an illuminating light beam is generally carried out with the aid of a controllable beam deflector Tilting two mirrors, moved in a sample plane, the Deflection axes are usually perpendicular to each other, so that a mirror in x, the other deflects in the y-direction.
  • the tilt of the mirror will accomplished for example with the help of galvanometer actuators.
  • the power of the detection light coming from the object is dependent measured from the position of the scanning beam.
  • the Control elements with sensors to determine the current mirror position equipped.
  • a confocal Scanning microscope generally includes a light source, a Focusing optics with which the light from the source is directed onto a pinhole diaphragm - the so-called. Excitation diaphragm - is focused, a beam splitter, a Beam deflection device for beam control, a microscope optics, a Detection aperture and the detectors for the detection of the detection resp. Fluorescent light. The illuminating light is transmitted through a beam splitter coupled.
  • This detection arrangement is Called the Descan arrangement. Detection light that does not come directly from the Focal region originates, takes a different light path and passes the Detection aperture not, so that you get a point information that through sequential scanning of the object with the focus of the Illuminating light beam leads to a three-dimensional image. Usually will a three-dimensional image is obtained by taking image data in layers.
  • Commercial scanning microscopes usually consist of a scanning module that is flanged to the stand of a classic light microscope, whereby the Scan module all of the above are additionally required for scanning a sample Includes elements.
  • a well-known method of fluctuations and fluctuations in the Compensating or correcting the illumination light output is based on a measuring beam from the illuminating light beam with the aid of a beam splitter and for image generation or image calculation the ratio of the measured powers of the measuring beam and detection light beam use. This procedure is for example in the publication G.J. Brakenhoff, Journal of Microscopy, Vol. 117, Pt 2, November 1979, pp. 233-242 disclosed.
  • the invention is therefore based on the object of a device to propose a largely accurate and reproducible determination the light output of a light beam allows.
  • a device which is characterized in that in the beam path of the measuring light a scattering optical element, such as a roughened glass plate or a Frosted glass pane, is provided so that the detector can use the measuring light receives reduced spatial and / or temporal coherence.
  • a scattering optical element such as a roughened glass plate or a Frosted glass pane
  • a microscope according to claim 8 which is characterized is that the beam splitter (1) and the detector (11) become one unit are summarized and that a in the beam path of the measuring light scattering optical element, such as a roughened glass plate or a Frosted glass pane, is provided so that the detector can use the measuring light receives reduced spatial and / or temporal coherence.
  • the measuring light scattering optical element such as a roughened glass plate or a Frosted glass pane
  • the invention has the advantage that a reliable measurement of the Light output of a light beam is made possible. Furthermore, the invention the advantage of a reliable, failure-prone and largely precise compensation of light output fluctuations, in particular of Fluctuations in the illuminating light for illuminating a sample in the Microscopy and scanning microscopy.
  • the detector also receives the measurement light reduced spatial and / or temporal coherence.
  • the Beam path of the measuring light a scattering optical element, such as a roughened glass plate or a frosted glass pane is provided.
  • the beam splitter comprises a Substrate that has a diffusely scattering surface, or at least that partly made of frosted glass.
  • An embodiment variant in which the detector is particularly advantageous is arranged immediately behind the beam splitter.
  • This variant is particularly not susceptible to external influences, not susceptible to interference and compact. Often multiple reflections in the beam splitter produce several susceptible to interference Partial beams, especially on the coated and uncoated ones Interfaces.
  • the detector is therefore advantageously arranged in such a way that that it is only from the primarily divided measurement light, but not from others Partial light rays, is illuminated.
  • the entrance window of the detector itself which is preferably is coated partially reflective, as a beam splitter.
  • the beam splitter consists of one Substrate that has a partially reflective coating.
  • it is a metallic or dielectric coating.
  • the coating is directly on the Detector or applied to the entrance window of the detector housing.
  • the Beam splitter or the coating is preferred in one Design variant carried out such that the ratio of Light output of the light beam compared to that measured at the detector Light output of the measuring light largely independent of the wavelength of the light beam is.
  • the beam splitter (1) and the detector (11) combined into a unit which has a housing.
  • An embodiment with a beam splitter is very particularly preferred, which generates a transmitted and a reflected partial beam and only the transmitted partial beam as a measuring beam on the detector meets.
  • the reflected partial beam is used as an illuminating light beam on a sample steered.
  • the microscope is a scanning microscope or a confocal scanning microscope, which preferably has a processing unit, the the measured power of the measuring light taking into account the Splitting ratio of the beam splitter and other system parameters with the Power of the detection light or a part, for example the power a part of the detection light from a certain spectral range, offset.
  • a processing unit is provided that a programmable digital electronics, for example FPGA (Field Programmable Gate Arrray).
  • A is preferably used to determine the power of the measuring light
  • Semiconductor detector such as a photodiode or avalanche or PIN diode CCD chip or a photodetector is provided, as semiconductor detectors are a have a particularly small design. Also photomultiplier or Photomultiplier arrays can be used.
  • Offsets can come from the detectors, for example if the zero adjustment is incorrect or they can be caused by scatter or Ambient light that inadvertently reaches the detectors.
  • the offsets are in a special form of a Scanning microscope automatically determined before scanning an image. For this purpose, the illuminating light is interrupted and that from the detectors outgoing signals measured and stored.
  • Scaling step which in the simplest case is multiplication by a Including constants, the corrected light output is applied to any scale customizable.
  • the method according to the invention comprises in the first two steps Determining a light output of an illuminating light beam with a Device comprising a beam splitter (1) and a beam splitter (1) associated detector (11), the beam splitter (1) measuring light (23) splits off from the illuminating light beam (37) and feeds it to the detector (11) and the ratio of the light output of the illuminating light beam (37) to the the light power of the measuring light (23) measured at the detector (11) over time is constant, and determining the light output of one from a sample outgoing detection light beam.
  • both Light outputs obtained with the help of detectors that correspond to the respective Generate electrical signals proportional to light output.
  • the signals are digitized in one embodiment and in an FPGA unit or in one PC offset against each other and thus a corrected detection light output determined which can be used for image generation or for image calculation.
  • the signals are offset in an analog manner.
  • the light output is determined of the illuminating light beam and the detection light beam simultaneously.
  • Fig. 1 shows a device for light power measurement according to the prior art Technology.
  • the device comprises a beam splitter 1, which consists of a substrate 3 with a partially reflective coating 5 consists.
  • the incident light beam 7 hits the coating 5 by reflection
  • Measuring light 9 split off which on the at a distance of about 3 cm arranged detector 11 meets.
  • the division ratio is approx. 50: 1.
  • Another measuring light 15 the power of which is approx. 4% of the power of the part of the light beam 7 which has passed through the coating is on the interface 13 to the detector 11 is reflected.
  • the coating split-off measuring light 9 interferes at the detector with that at the measurement light split off from the uncoated interface 13 of the substrate 3 15. Even the slightest variations in the optical path length between the Beam splitter 1 and the detector 11 and in the substrate 3, for example through Changes in temperature, air pressure fluctuations or vibrations lead to a change in interference resulting in a large variation in the measured light output is reflected.
  • Fig. 2 shows a device according to the invention for determining the Light output of a light beam 7.
  • the device comprises a beam splitter 1 with a substrate 17 which has a smooth surface with a partially transparent, metallic coating 19 and a roughened, diffuse having scattering surface 21.
  • a detector 11 which is designed as a photodiode, puttied up.
  • the light beam 7 striking the beam splitter 1 is shown in FIG transmitted measuring light 23 and a reflected partial beam 25 in relation 1:40 shared. Due to the roughened surface 21, the interference ability of the the detector 11 incident light largely destroyed.
  • the device is on Due to its small size, it is extremely immune to external interference.
  • Of the Detector generates a signal for the output of the incident measuring light 23 proportional electrical signal that is transmitted via line 27 of a Processing unit can be fed
  • Fig. 3 shows another device according to the invention for determining the Light output of a light beam.
  • the device includes a beam splitter 1, which has a substrate made of frosted glass 29 and a partially reflective, dielectric Coating 31. Has. The light beam 7 striking the coating is reflected to 97%. The measuring light 23 passing through the coating becomes sprinkled in the substrate of milk glass 29 and reaches the detector 11 with largely destroyed interference ability. The detector generates a for Power of the incident measuring light 23 proportional electrical signal, which can be fed to a processing unit via line 27.
  • Fig. 4 shows schematically a microscope 33 according to the invention, which as confocal scanning microscope is carried out.
  • the one from a lighting system 35 incoming illuminating light beam 37 is transmitted via a glass fiber 39 transported and hits after decoupling from the glass fiber 39 with the help of Optics 41 to a device 43 for determining the performance of the Illuminating light beam emitting a measuring beam with a beam splitter 1 divides the illuminating light beam and supplies it to the detector 11.
  • the Beam splitter 1 includes a substrate made of milk glass.
  • the detector 11 generates an electrical signal proportional to the power of the measuring light, which is transmitted via the line 45 is passed to the processing unit 47.
  • the illuminating light beam 37 reaches the gimbal suspended scanning mirror 51, the beam through the scanning optics 53, the Tube optics 55 and the objective 57 through over or through the sample 59 leads.
  • the illuminating light beam 37 is 59 in the case of non-transparent samples guided over the sample surface.
  • the illuminating light beam 37 can also pass through transparent samples the sample 59 are guided. This means that from different Focal planes of the object one after the other through the illuminating light beam 37 are scanned. The subsequent composition then results in a three-dimensional image of the sample.
  • the one emanating from the sample Detection light 61 passes through the objective 57, the tube optics 55 and the Scan optics 53 through and via the scan mirror 51 to the beam splitter 49, passes this and meets a detector device 63, which as Photomultiplier is executed.
  • a detector device 63 which as Photomultiplier is executed.
  • electrical electrical proportional to the output of the detection light Detection signals are generated and sent via line 65 to the processing unit 47 passed.
  • the processing unit 47 the incoming, Analog signals are first digitized and then digitally offset against each other and determine a corrected detection light output. This is sent to a PC 67 passed.
  • the corrected detection light output is based on a Position signal of the gimbaled mirror the position of the associated grid point and the data of all grid points to composed of an image of the sample 69, which is shown on a display 71 is shown.
  • This is usually the case with a confocal scanning microscope provided illumination pinhole 73 and the detection pinhole 75 are the Drawn in schematically for the sake of completeness. Are omitted because of for better clarity, on the other hand, some optical elements for guidance and shaping the light rays. These are one active in this field Well known to those skilled in the art.
  • Fig. 5 shows schematically a further microscope 33 according to the invention, the is designed as a confocal scanning microscope.
  • the illustrated microscope 33 corresponds largely to the microscope shown in FIG.
  • the device 43 to determine the power of the illuminating light beam is in this Execution between the illumination pinhole 73 and the beam splitter 49 arranged.
  • FIG. 6 shows schematically a further microscope 33 according to the invention, which is designed as a confocal scanning microscope.
  • the illustrated microscope 33 corresponds largely to the microscope shown in FIG.
  • the device 43 to determine the power of the illuminating light beam is in this Execution between the gimbal-mounted scanning mirror 51 and the Scanning optics 53 arranged.
  • the microscope 33 has the additional advantage that also fluctuations in the power of a polarized Illuminating light beam 37 are detected because of different Reflectances of the beam splitter 49 for different Directions of polarization, for fluctuations in the direction of polarization are due.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)
  • Mechanical Optical Scanning Systems (AREA)

Claims (19)

  1. Dispositif pour déterminer la puissance lumineuse d'un faisceau lumineux (7) avec un séparateur de faisceau (1) et un détecteur (11) associé au séparateur de faisceau (1), le séparateur de faisceau (1) séparant une lumière de mesure (23) du faisceau lumineux (7) et la dirigeant vers le détecteur (11), et le séparateur de faisceau (1) et le détecteur (11) étant réunis en une unité, caractérisé en ce qu'un élément optique diffusif, comme une plaque de verre (21) rendue rugueuse ou un disque à verre dépoli (29), est prévu dans la trajectoire du faisceau de la lumière de mesure (23) pour que le détecteur (11) reçoive la lumière de mesure (23) avec une cohérence spatiale et/ou temporelle diminuée.
  2. Dispositif selon la revendication 1, caractérisé en ce que le séparateur de faisceau (1) comprend un substrat (17, 29) qui présente une surface (21) diffusive ou se compose au moins partiellement de verre dépoli (29).
  3. Dispositif selon l'une quelconque des revendications 1 ou 2, caractérisé en ce que le détecteur (11) est disposé directement derrière le séparateur de faisceau (1).
  4. Dispositif selon l'une quelconque des revendications 2 ou 3, caractérisé en ce que le séparateur de faisceau (1) présente un revêtement (19, 31) qui est appliqué sur le substrat (17, 29).
  5. Dispositif selon la revendication 4, caractérisé en ce que le revêtement (19, 31) est un revêtement métallique.
  6. Dispositif selon l'une quelconque des revendications 1 à 5, caractérisé en ce que le séparateur de faisceau (1) produit une lumière de mesure transmise (79) et un faisceau partiel réfléchi (25) et la lumière de mesure transmise (79) est exclusivement incidente sur le détecteur (11).
  7. Dispositif selon la revendication 1, caractérisé en ce que le séparateur de faisceau (1) est formé de sorte que le rapport des puissances lumineuses du faisceau lumineux (7) et de la lumière de mesure (23) est indépendant de la longueur d'onde du faisceau lumineux (7).
  8. Microscope (33) avec une source lumineuse, qui émet un faisceau lumineux d'illumination (37) pour illuminer un échantillon (59), avec au moins un dispositif détecteur (63) pour détecter la lumière de détection (61) sortant de l'échantillon (59) et un dispositif pour déterminer la puissance lumineuse d'un faisceau lumineux d'illumination (37) avec un séparateur de faisceau (1) et un détecteur (11) associé au séparateur de faisceau (1), le séparateur de faisceau (1) séparant une lumière de mesure (23) du faisceau lumineux d'illumination (37) et la dirigeant vers le détecteur (11), caractérisé en ce que le séparateur de faisceau (1) et le détecteur (11) sont réunis en une unité et qu'un élément optique diffusif, comme une plaque de verre (21) rendue rugueuse ou un disque à verre dépoli (29), est prévu dans la trajectoire de faisceau de la lumière de mesure (23) pour que le détecteur (11) reçoive la lumière de mesure (23) avec une cohérence spatiale et/ou temporelle diminuée.
  9. Microscope (33) selon la revendication 8, caractérisé en ce que le séparateur de faisceau (1) comprend un substrat (17, 29) qui présente une surface (21) diffusive ou se compose au moins partiellement de verre dépoli (29).
  10. Microscope (33) selon l'une quelconque des revendications 8 ou 9, caractérisé en ce que l'unité présente un boítier.
  11. Microscope (33) selon l'une quelconque des revendications 8 à 10, caractérisé en ce que le détecteur (11) est disposé directement derrière le séparateur de faisceau (1).
  12. Microscope (33) selon l'une quelconque des revendications 9 à 11, caractérisé en ce que le séparateur de faisceau (1) présente un revêtement (19, 31) qui est appliqué sur le substrat (17, 29).
  13. Microscope (33) selon la revendication 8, caractérisé en ce que le rapport des puissances lumineuses du faisceau lumineux d'illumination (37) et de la lumière de mesure (23) est indépendant de la longueur d'onde du faisceau lumineux d'illumination (37).
  14. Microscope (33) selon l'une quelconque des revendications 8 à 13, caractérisé en ce que le microscope (33) est un microscope à balayage ou un microscope à balayage confocal.
  15. Procédé pour la microscopie caractérisé par les étapes suivantes :
    détermination d'une puissance lumineuse d'un faisceau lumineux d'illumination (37) avec un dispositif, qui comprend un séparateur de faisceau (1) et un détecteur (11) associé au séparateur de faisceau (1), le séparateur de faisceau (1) séparant une lumière de mesure (23) du faisceau lumineux d'illumination (37) et la dirigeant vers le détecteur (11) et un élément optique diffusif, comme une plaque de verre (21) rendue rugueuse ou un disque à verre dépoli (29), étant prévu dans la trajectoire de faisceau de la lumière de mesure (23) pour que le détecteur (11) reçoive la lumière de mesure (23) avec une cohérence spatiale et/ou temporelle diminuée,
    détermination d'une puissance lumineuse d'un faisceau lumineux de détection sortant d'un échantillon (59) et
    détermination d'une puissance lumineuse corrigée par calcul de la puissance lumineuse du faisceau lumineux d'illumination (37) et de la puissance lumineuse du faisceau lumineux de détection.
  16. Procédé selon la revendication 15, caractérisé en ce que la détermination de la puissance lumineuse du faisceau lumineux d'illumination (37) et du faisceau lumineux de détection se réalise simultanément.
  17. Procédé selon la revendication 15, caractérisé en ce que la détermination de la puissance lumineuse du faisceau lumineux d'illumination (37) comprend un ajustement de décalage.
  18. Procédé selon la revendication 15, caractérisé en ce que la détermination de la puissance lumineuse du faisceau lumineux de détection comprend un ajustement de décalage.
  19. Procédé selon l'une quelconque des revendications 15 à 18, caractérisé en ce que la détermination comprend une étape de mise à l'échelle.
EP02100561A 2001-05-25 2002-05-24 Dispositif pour la détermination d'une efficacité lumineuse, microscope et méthode de microscopie Expired - Lifetime EP1260848B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10125469A DE10125469B4 (de) 2001-05-25 2001-05-25 Vorrichtung zur Ermittlung einer Lichtleistung, Mikroskop und Verfahren zur Mikroskopie
DE10125469 2001-05-25

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EP1260848A2 EP1260848A2 (fr) 2002-11-27
EP1260848A3 EP1260848A3 (fr) 2002-12-18
EP1260848B1 true EP1260848B1 (fr) 2005-07-20

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US (1) US6806950B2 (fr)
EP (1) EP1260848B1 (fr)
JP (1) JP2003075764A (fr)
DE (2) DE10125469B4 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016122529A1 (de) 2016-11-22 2018-05-24 Carl Zeiss Microscopy Gmbh Mikroskop zur Abbildung eines Objekts
DE102016122528A1 (de) 2016-11-22 2018-05-24 Carl Zeiss Microscopy Gmbh Verfahren zum Steuern oder Regeln einer Mikroskopbeleuchtung
WO2018096003A1 (fr) 2016-11-22 2018-05-31 Carl Zeiss Microscopy Gmbh Microscope pour imager un objet
WO2018096004A1 (fr) 2016-11-22 2018-05-31 Carl Zeiss Microscopy Gmbh Procédé de commande ou de réglage d'un éclairage de microscope
CN110036328A (zh) * 2016-11-22 2019-07-19 卡尔蔡司显微镜有限责任公司 用于控制或调节显微镜照明的方法
US10823948B2 (en) 2016-11-22 2020-11-03 Carl Zeiss Microscopy Gmbh Microscope for imaging an object
US11774739B2 (en) 2016-11-22 2023-10-03 Carl Zeiss Microscopy Gmbh Method for controlling or regulating a microscope illumination

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EP1260848A3 (fr) 2002-12-18
DE10125469B4 (de) 2008-01-10
JP2003075764A (ja) 2003-03-12
DE50203658D1 (de) 2005-08-25
US20020191177A1 (en) 2002-12-19
US6806950B2 (en) 2004-10-19
EP1260848A2 (fr) 2002-11-27
DE10125469A1 (de) 2002-12-12

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